Abstract The recent FDA approval of two more antibody drug conjugates (ADCs) highlights the clinical success and growth in this class of agents. Despite these approvals, however, the attrition rate for new ADCs remains high, and in oncology applications, no ADC for solid tumors has yet been able to repeat the success of ado- trastuzumab emtansine (T-DM1). A substantial effort has been exerted to improve the antibody and target selection, conjugation site, linker stability, and payload properties (potency, bystander effects, etc.), and these improvements will benefit the next generation of compounds. However, fundamental questions remain on how to design a clinically effective agent. Specifically, the relative contribution and interaction between the multiple mechanisms of action of these drugs (receptor signaling blockade, payload efficacy, and Fc effector functions) remains unknown. The long-term goal is to understand the fundamental properties of these complex drugs in sufficient detail to rationally combine the antibody, linker, and payload with a particular target (in a select patient population) for maximum clinical efficacy in both cancer and non-oncologic applications. The goal for this proposal is to quantitatively understand the relative contribution of direct payload effects in antigen positive cells, bystander payload effects in antigen negative cells, and the role of Fc-effector functions in determining efficacy with antibody drug conjugates. Using a combination of near-infrared fluorescence imaging and flow cytometry, the absolute number of intact and degraded (triggering payload release) ADCs per cell can be determined. By pairing these results with pharmacodynamic markers (e.g. DNA damage markers of alkylating agents), the delivery and efficacy of the ADC (both direct and bystander killing) can be quantified with single cell resolution in vivo. Co-administration of varying ratios of ADC with unconjugated antibody will be used to control the tissue distribution to vary direct versus indirect killing. The studies will be conducted in both an immunocompromised and immunocompetent (syngeneic) mouse model to determine the benefit (or requirement) for immune system activation. By comparing the single-cell measurements with the gold standard of preclinical efficacy (tumor growth curves), the relative contribution of each mechanism will be determined. The outcome of the work will enable the rational design of novel ADCs rather than testing the myriad combinations in vivo by focusing development on a) selection of targets with more uniform expression and ADC internalization if direct targeting predominates, b) optimal physicochemical properties and distribution of bystander payloads if bystander effects plays a major role, or c) activation and recruitment of immune cells through co-therapy, Fc engineering, and/or dosing regimens if immune cell recruitment is necessary. A significant number of monoclonal antibodies that failed as monotherapies are sitting dormant within pharmaceutical companies. By leveraging advances in payload and linker chemistry with the knowledge from this proposal, development of new clinically successful ADCs can be rapidly accelerated.